PROJECT SUMMARY Alzheimer?s disease (AD) is among the leading causes of a poor life quality and death in the elderly. No treatment available stops the progression of AD because of poor understanding of mechanisms underlying this disease. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the translation of targeted mRNAs and play important roles in almost every critical cellular process. Also, multiple lines of evidence indicate that miRNAs are essential for neuron function and survival. Not surprisingly, alterations of individual miRNAs have been implicated in the AD pathological condition. The number of miRNAs is dysregulated in the AD conditions. Several neuroprotective miRNAs are markedly downregulated, while several neurodegenerative miRNAs are significantly upregulated in the same AD context. Identifying what reason causes the differentiative expressions of miRNAs may be key to the understanding of AD pathogenesis. MiRNA biogenesis is controlled by several tightly coupled sequential steps. What step of biogenesis is dysregulated under AD conditions? Our preliminary data have revealed that dysregulation of key AD-associated miRNAs occurs at their step from primary miRNAs (pri-miRNAs) to precursor miRNAs (pre-miRNAs). Nuclear RNase III enzyme Drosha with its partner DGCR8 forms the microprocessor complex and acts first in the cascade to process pri-miRNA transcripts into pre-miRNA transcripts. The microprocessor complex is highly regulated, but its regulation and mechanisms underlying individual miRNAs are poorly understood. In this project, based on our preliminary findings, we can confidently create a hypothesis that the altered proteins in individual miRNA microprocessor complexes by AD stress deregulate subset miRNA biogenesis and cause AD- associated pathological changes. We propose the following specific aims to test this hypothesis: Aim I: Determine how AD-associated miRNAs change at pri-, pre-, and mature levels in three level conditions; Aim II: Develop novel specific probe-based proteomics techniques to dissect differentiations of protein components; and Aim III: Identify altered proteins in microprocessor complex serving as regulators for miRNA biogenesis and determining the effect of intervention of regulators on AD-associated pathology. How AD-associated stress dysregulates microprocessor components may be crucial to understanding the neurodegenerative process in AD. A positive outcome of the proposed study will reveal biogenetical mechanisms underlying individual AD-associated miRNAs. Revealed novel regulators should serve as more effective therapeutic targets to address AD pathological processes. The findings will have a high impact on our understanding of the degenerative process of AD and possibly other neurodegenerative diseases.